An efficient mammalian cell-free translation system supplemented with translation factors
An efficient mammalian cell-free translation system supplemented with translation factors
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DOI：
10.1016/j.pep.2005.09.021
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发表时间：
2006-04-01
影响因子：
1.6
通讯作者：
Imataka, H
中科院分区：
文献类型：
--
作者：
Mikami, S;Masutani, M;Imataka, H
从哺乳动物细胞中开发高效的无细胞翻译系统是一个重要的目标。我们研究了HeLa细胞提取物中添加任何翻译起始因子或翻译调节因子是否可以促进蛋白质合成。eIF2（真核生物翻译起始因子2）和eIF2B增强了带帽、未带帽和脑心肌炎病毒-内部核糖体进入位点促进mrna的翻译。eIF4E特异性地刺激了带帽mRNA的翻译，而p97 （eIF4G c端三分之二的同源物）增加了未带帽mRNA的翻译。当HeLa细胞提取物中添加eIF2、eIF2B和p97的组合时，从未封盖的mRNA合成蛋白质的能力与由eIF2、eIF2B和eIF4E组合刺激的封盖对应物的能力相当。透析法使HeLa细胞提取物能够在36小时内合成蛋白质，并且当补充起始因子时产量增加。相比之下，这种方法并没有提高兔网织细胞裂解液的生产效率。总之，翻译因子补充的HeLa细胞提取物应该成为生产重组蛋白的重要工具。(c) 2005爱思唯尔公司版权所有。
Development of an efficient cell-free translation system from mammalian cells is an important goal. We examined whether supplementation of HeLa cell extracts with any translation initiation factor or translational regulator could enhance protein synthesis. eIF2 (eukaryotic translation initiation factor 2) and eIF2B augmented translation of capped, uncapped and encephalomyocarditis virus-internal ribosome entry site-promoted mRNAs. eIF4E specifically stimulated capped mRNA translation, while p97, a homologue to the C-terminal two-thirds of eIF4G, increased uncapped mRNA translation. When the HeLa cell extract was supplemented with a combination of eIF2, eIF2B, and p97, the capacity to synthesize a protein from an uncapped mRNA became comparable to that from the capped counterpart stimulated with a combination of eIF2, eIF2B, and eIF4E. A dialysis method rendered the HeLa cell extract capable of synthesizing proteins for 36 h, and the yield was augmented when supplemented with initiation factors. In contrast, the productivity of a rabbit reticulocyte lysate was not enhanced by this method. Collectively, the translation factor-supplemented HeLa cell extract should become an important tool for the production of recombinant proteins. (c) 2005 Elsevier Inc. All rights reserved.